Prostate cancer is the most commonly diagnosed malignancy for men in the United States with an estimated 238,590 new cases projected for 2013. The most current means for detecting prostate cancer is a combination of a digital rectal exam (DRE) and monitoring levels of prostate-specific antigen (PSA) in the blood. Prostate-specific antigen is a protease produced by the prostate gland. PSA is present at low concentration in the blood of healthy males, and an increase in the concentration of PSA in the blood can be indicative of a prostate tumor. Until recently, PSA testing was recommended as a screening tool for all men over 50. However, two large-scale, randomized trials of PSA screening suggest that prostate cancer is over-diagnosed and over-treated, likely because many cancers that are detected are never destined to progress. Prostate cancer can have an aggressive and lethal course and an estimated 29,720 men are projected to die of prostate cancer in 2013, however, for most patients, prostate cancer is a slow growing disease. This broad range of clinical behavior is likely a reflection of the underlying genomic diversity of the tumors. Previous studies of prostate tumors reported significant heterogeneity in the gene expression profiles and genomic structural alterations including DNA copy number changes and gene fusions often involving the ETS family of transcription factors detectable in approximately half of prostate tumors. Exon sequencing of known oncogenes and tumor suppressors has found few somatic mutations and the calculated background mutation rate appears to be relatively low. This suggests the presence of other forms of genomic aberrations that contribute to the observed gene expression variations, and in turn, the diversity in tumor behavior.
Methods of detecting and/or diagnosing prostate cancer have been described previously. See for instance the following issued U.S. Pat. No. 7,524,633—Method of detection of prostate cancer; U.S. Pat. No. 7,427,476—PITX2 polynucleotide, polypeptide and methods of use therefore; U.S. Pat. No. 7,381,808 Method and nucleic acids for the differentiation of prostate tumors; U.S. Pat. No. 7,252,935—Method of detection of prostate cancer; U.S. Pat. No. 7,195,870—Diagnosis of diseases associated with gene regulation; U.S. Pat. No. 7,049,062—Assay for methylation in the GST-Pi gene; U.S. Pat. No. 6,864,093—Method of identifying and treating invasive carcinomas; U.S. Pat. No. 6,815,166—HIN-1, a tumor suppressor gene; U.S. Pat. No. 6,783,933—CACNA1G polynucleotide, polypeptide and methods of use therefore; U.S. Pat. No. 6,569,684—Method of identifying and treating invasive carcinomas; U.S. Pat. No. 5,552,277—Genetic diagnosis of prostate cancer; and U.S. Pat. No. 5,846,712 Tumor suppressor gene, HIC-1. In addition, conventional methods utilize the prostate specific antigen (PSA) blood test, and the digital rectal exam (DRE). PSA is an enzyme produced in the prostate that is found in the seminal fluid and the bloodstream. An elevated PSA level in the bloodstream does not necessarily indicate prostate cancer, since PSA can also be raised by infection or other prostate conditions such as benign prostatic hyperplasia (BPH). Many men with an elevated PSA do not have prostate cancer. Nonetheless, a PSA level greater than 4.0 nanograms per milliliter of serum was established initially as the cutoff where the sensitivity for detecting prostate cancer was the highest and the specificity for detecting non-cancerous conditions was the lowest. A PSA level above 4.0 ng per milliliter of serum may trigger a prostate biopsy to search for cancer. The digital rectal exam is usually performed along with the PSA test, to check for physical abnormalities that can result from tumor growth.
The PSA test is an imperfect screening tool. A man can have prostate cancer and still have a PSA level in the “normal” range. Approximately 25% of men who are diagnosed with prostate cancer have a PSA level below 4.0. In addition, only 25% of men with a PSA level of 4-10 are found to have prostate cancer. With a PSA level exceeding 10, this rate jumps to approximately 65%.
Current diagnostic tools for prostate cancer lack the sensitivity and specificity required for the detection of very early prostate lesions and diagnosis ultimately relies on an invasive biopsy. Once prostate cancer is diagnosed, there are no available prognostic markers for prostate cancer that provide information on how aggressively the tumor will grow. Therefore, more intrusive therapeutic routes are often chosen that result in a drastic reduction in the quality of life for the patient, even though the majority of prostate tumors are slow growing and non-aggressive. This ultimately leads to undue burden on the healthcare system and an unnecessary decrease in quality of life for the patient. The present invention addresses the need for distinguishing aggressive prostate tumors through identification of specific genomic DNA methylation biomarkers that can distinguish patients that will undergo biochemical recurrence.
DNA methyltransferases (also referred to as DNA methylases) transfer methyl groups from the universal methyl donor S-adenosyl methionine to specific sites on a DNA molecule. Several biological functions have been attributed to the methylated bases in DNA, such as the protection of the DNA from digestion by restriction enzymes in prokaryotic cells. In eukaryotic cells, DNA methylation is an epigenetic method of altering DNA that influences gene expression, for example during embryogenesis and cellular differentiation. The most common type of DNA methylation in eukaryotic cells is the methylation of cytosine residues that are 5′ neighbors of guanine (“CU” dinucleotides, also referred to as “CpGs”). DNA methylation regulates biological processes without altering genomic sequence. DNA methylation regulates gene expression, DNA-protein interactions, cellular differentiation, suppresses transposable elements, and X Chomosome inactivation.
Improper methylation of DNA is believed to be the cause of some diseases such as Beckwith-Wiedemann syndrome and Prader-Willi syndrome. It has also been purposed that improper methylation is a contributing factor in many cancers. For example, de novo methylation of the Rb gene has been demonstrated in retinoblastomas. In addition, expression of tumor suppressor genes have been shown to be abolished by de novo DNA methylation of a normally unmethylated 5′ CpG island. Many additional effects of methylation are discussed in detail in published International Patent Publication No. WO 00/051639.
Methylation of cytosines at their carbon-5 position plays an important role both during development and in tumorigenesis. Recent work has shown that the gene silencing effect of methylated regions is accomplished through the interaction of methylcytosine binding proteins with other structural components of chromatin, which, in turn, makes the DNA inaccessible to transcription factors through histone deacetylation and chromatin structure changes. The methylation occurs almost exclusively in CpG dinucleotides. While the bulk of human genomic DNA is depleted in CpG sites, there are CpG-rich stretches, so-called CpG islands, which are located in promoter regions of more than 70% of all known human genes. In normal cells, CpG islands are unmethylated, reflecting a transcriptionally active state of the respective gene. Epigenetic silencing of tumor suppressor genes by hypermethylation of CpG islands is a very early and stable characteristic of tumorigenesis. Hypermethylation of CpG islands located in the promoter regions of tumor suppressor genes are now firmly established as the most frequent mechanisms for gene inactivation in cancers.